The rapid development of biotechnology depends heavily on engineered microbes. In particular, the booming field of synthetic biology demonstrates the feasibility of de novo synthesis of viral genomes, bacterial genomes and eukaryotic chromosomes. The technologies underpinning synthetic biology are advanced DNA synthesis and assembly, genome editing and computational assisted designs, which are all becoming commoditized and thus increasingly available to the public. The advance of synthetic biology promises to ultimately improve human living conditions through a better understanding of fundamental sciences as well as a multitude of practical applications. However biosafety mechanisms should be carefully considered to minimize or prevent dual use. Professionals have chemically synthesized infectious virus in the absence of natural templates (Cello J, Paul A V, & Wimmer E (2002) Science 297(5583):1016-1018) and reconstitute infectious human retroviruses (Lee Y N & Bieniasz P D (2007) PLoS pathogens 3(1):e10.) and the 1918 ‘Spanish’ influenza virus (Tumpey T M, et al. (2005) Science 310(5745):77-80). At the same time, relative amateurs are trying to engineer microbes in a Do-It-Yourself fashion (www.diybio.org). Proactive measures are warranted to minimize both bioterror (e.g. the anthrax attack the United States in 2000) and “bioerror” such as accidental environment releases or self-infection by lab-adapted microbes as in the case of a lab infection of an individual with hemochromatosis, where the victim scientist's high iron levels caused by hemochromatosis complemented the natural iron requirement of attenuated Y. pestis (Frank K M, et. (2011) The New England journal of medicine 364(26):2563-2564). Intrinsic biocontainment can also be used to prevent industrial espionage.
Work on biocontainment has largely focused on auxotrophic mutations or inducible lethality based on toxin-antitoxin pairs in bacteria (Bej A K, et al (1988) Applied and environmental microbiology 54(10):2472-2477; Gerdes K, et al. (1986) The EMBO journal 5(8):2023-2029; Knudsen S M & Karlstrom O H (1991) Applied and environmental microbiology 57(1):85-92; Poulsen L K, et al. Molecular microbiology 3(11):1463-1472). There is also recent work to reduce unintended plasmid propagation in bacterial (Wright O, et al., (2014) GeneGuard: A Modular Plasmid System Designed for Biosafety. ACS synthetic biology). Existing biocontainment technologies usually depend on a single cellular mechanism. Such “uniplex” approaches lack redundancy, and the leakiness of toxin genes often compromises fitness and normal behavior of the engineered microbes, reducing the likelihood that they will be accepted by researchers or industrial biotechnologists. Also, ongoing selection for the toxin gene is required. Reduced fitness has at least three undesirable features: 1) it reduces their usefulness as models for the behavior of the natural organism 2) it may reduce their “performance” in industrial applications and 3) it may increase the frequency of escape mutants (revertants able to grow in the absence of the compound). Finally, auxotrophic strains rely on supplementation of corresponding nutrients at micromolar concentrations, rendering them too costly for industrial scale-up. Thus there is an ongoing need for improved compositions and methods designed for biocontainment of microorganisms. The present disclosure meets these and other needs.